Amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease) is an age-dependent degenerative disorder of motor neurons characterized by progressive weakness and death within five years after the onset of symptoms. A subset of familial ALS is caused by mutations in Cu/Zn superoxide dismutase (SOD1) that render this antioxidant enzyme toxic by an unknown mechanism. Expression of mutant SOD1 in man or mice causes selective degeneration of motor neurons in the brain and spinal cord. The long-term objectives of this study are to understand how mutant SOD1 kills neurons and to identify novel pathways of cell death that may be relevant to ALS. Aim 1 of this project is to define important biophysical and biochemical characteristics that distinguish mutant from wild type SOD1. Recent studies in our lab indicate that mutant SOD1 enzymes all share an increased tendency to unfold or to lose metal ions when stressed by denaturing influences. Experiments will be performed to (1) define conditions that preferentially destabilize mutant but not normal SOD1, (2) identify factors that alter the dynamics of the SOD1 monomer-dimer equilibrium as measured by analytical ultracentrifugation, and (3) map regions of the protein that are susceptible to proteolytic cleavage or partial unfolding. In Aim 2, the affinities and thermodynamics of metal binding to SOD1 as a function of pH or denaturant will be measured using a novel calorimetric approach. Conditions that alter Cu ion reactivity in mutant SOD1 will also be defined. Aim 3 will employ cell culture models to test hypotheses of mutant SOD1 toxicity that are consistent with the physicochemical findings from Aims 1 and 2. Initial investigations will test whether early lysosomal dysfunction occurs in response to specific stresses in mutant SOD1 -bearing neuroblastoma cells or in organotypic spinal cord slice cultures. Future results from Aims 1 and 2 may favor alternative hypotheses of mutant SOD1 toxicity to test in these cellular models and may suggest new therapeutic approaches to evaluate in ALS mice.